Adhesion inhibiting composition

ABSTRACT

An extract prepared from plants of the genus Vaccinium, especially cranberries, which is enriched for an activity which inhibits bacterial adhesion to surfaces. The extract is also enriched for polyphenol and flavonoid compounds, lacks detectable amounts of simple sugars, has a very low content of benzoic acid relative to raw cranberries, and lacks significant amounts of anthocyanins. Methods for preparing and for using the extract are also disclosed.

BACKGROUND OF THE INVENTION

1. Field

The invention relates to plant extracts having therapeutic and otheruses, and more particularly to an extract of cranberries.

2. State of the Art

It is presently believed that cranberry juice or some fraction thereofinhibits the adhesion of bacteria to mammalian cells, particularlyepithelial cells. This property of anti-adhesion can be applied toindustrial uses, such as the cleaning of vats from bacterialfermentation, cleaning of medical and dental instruments, cleaning ofdishes or tools used in sterile laboratory procedures, and the like.

Many persons also consider cranberry juices and cranberry derivatives tobe beneficial to health, and products including powders made fromcranberries or cranberry juices are available commercially. Doctorsoften recommend cranberry products for female patients suffering fromurinary tract infections. However, most available preparations, as wellas raw cranberries and typical cranberry juice products have arelatively high acidity due in part to the amount of benzoic acidpresent. The acidity can cause stomach upset, stimulate tooth decay, andproduces a sour taste which is unappealing to many persons.

Consequently, a need remains for a cranberry extract which includes theactive fraction of cranberries responsible for the anti-bacterialadhesion activity. A need also remains for a composition in which theactive fraction is concentrated and/or partially purified to have ahigher anti-adhesion activity per weight unit. It would furthermore behighly desirable to provide reliable markers for identifying the activefraction during a concentrating or purifying process. A need alsoremains for a cranberry extract which contains the active fraction buthas substantially reduced acidity and sugar content.

SUMMARY OF THE INVENTION

The invention comprises an extract made from plant material of plantspecies of the genus Vaccinium, and which is significantly enriched foran activity that interferes with adhesion of bacterial cells tosurfaces. The extract is also enriched for polyphenol and flavonoidcompounds including ones having a glycoside moiety. When the extract isanalyzed by reverse-phase HPLC on a C18 lipophilic column,characteristic sets of elution peaks of compounds absorbing at 230 nm,280 nm and 360 nm are observed. The extract may also contain tannincompounds represented by elution peaks absorbing in the ultravioletwavelength region.

In the presently preferred embodiment, the extract is enriched by atleast about 500- to 1500-fold for the anti-adhesion activity, ascompared with juices which are 100% derived from the plant material. Theextract is enriched to a similar degree in the concentration offlavonoid and other polyphenol compounds detected by spectroscopicmethods.

In a currently preferred embodiment, the extract is very low in acid andin simple sugars, with a benzoic acid content typically less than about0.01 milligrams per gram dry powder, and essentially undetectableamounts of free monomer or dimer sugars. The preferred embodiment isprepared from cranberries (V. macrocarpon and variants). However, otheruseful species are V. myrtilis (bilberry), V. oxycoccus (Europeancranberry), and V. corymbosum (blueberries).

The invention also includes a method of making an extract having theproperties outlined in the preceding paragraphs, and a method ofinhibiting the adhesion of bacteria to surfaces using the extract.

The presently preferred method of making the extract includes at leastthe steps of: preparing a starting extract from plants or plant parts ofspecies selected from the genus Vaccinium, this starting extractincluding charged and polar compounds and the active fraction;concentration of the extract to a smaller volume; and enrichment of theextract for the active fraction and for polyphenol and flavonoidcompounds generally. In a presently preferred embodiment, the methodincludes the further steps of: removing most of the free monomer anddimer sugars from the extract; removing most of the benzoic acid fromthe extract; removing anthocyanins. Steps following the preparation of astarting extract, are not necessarily performed in the order listed.Techniques are described for accomplishing each of the indicated stepsby chromatography or by precipitation and phase extraction steps.Additionally, in one embodiment the method includes a step of mannoseaffinity chromatography which selects for compounds that can compete forbinding to a mannose-affinic substrate.

The invention further embraces compositions produced by first extractingnon-active compounds from plant materials with a basic (high pH)solution, leaving a pulp or residue enriched for the anti-adhesionactive fraction. This pulp or residue may be further processed by acidsolubilization followed by selected steps as described in the precedingparagraphs, to further enrich for the active fraction.

The anti-adhesion property of the extract is useful in a number ofareas, for example the cleaning of industrial fermentation equipment,medical and dental instruments, laboratory culture jars, and the like.The extract may further have usefulness to inhibit the adhesion ofbacteria to surgical implants, to tooth surfaces and oral cell typesfound in the mouth, and to cells in the urinary tract of humans and/oranimals. The presently preferred extract has a greatly reduced contentof both acids and sugar, and is an excellent food supplement to replacecranberries and cranberry juice. The low sugar and acid content make theextract highly suitable for oral hygiene products, and more useful tothose who seek the health benefits of a cranberry extract product.

A method of inhibiting the adhesion of bacteria comprises the steps ofproviding an extract as described, applying the extract in a suitablemedium to a surface(s) having bacteria such as E. coli adhered thereto,to disengage the bacteria from the surface(s). The method is useful toinhibit the adhesion of bacteria to such surfaces as teeth, otherbacteria adhered to teeth, to human oral epithelial cells, humanepithelial urinary tract cells; and to clean dental implants, bacterialfermentation vats, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which illustrate what is presently regarded as thepreferred embodiment,

FIG. 1 is a flow chart illustrating the steps of preparation of anextract according to the invention;

FIG. 2 is an infrared absorbance spectrum of the extract dissolved inwater;

FIG. 3 is an ultra-violet/visible light absorbance spectrum of theextract dissolved in methanol;

FIG. 4 is an ultra-violet/visible light absorption spectrum of theextract dissolved in water;

FIGS. 5A-5B depicts a comparative analysis of a high-pressure liquidchromatogram of the extract by absorbance at wavelengths of 230 nm, 280nm, 360 nm and 512 nm;

FIGS. 6A-6B is a chart depicting a dual spectral analysis and analysisof elution peaks absorbing at 360 nm, of a high-pressure liquidchromatogram of the extract;

FIGS. 7A-7F contains charts depicting the complete UV-visible lightspectra of selected HPLC fractions taken from the sample whosechromatogram is shown in FIGS. 5A-5B;

FIG. 8 depicts a chromatogram of a first-step extract of crushedcranberries analyzed at multiple wavelengths;

FIG. 9 depicts a chromatogram of the extract obtained with a differentcolumn volume than the chromatograms of FIGS. 5A-5B and 6A-6B;

FIG. 10 is a flow chart depicting the steps of an alternate method forpreparation of an extract according to the invention;

FIGS. 11A-11F are HPLC chromatograms of products of selected steps inthe process of the invention;

FIG. 12 is a chromatogram of a product of step 1002 indicatingcollection of sequential fractions for activity analysis.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENT

An extract of species of the genus Vaccinium which is highly enrichedfor an active fraction having activity to inhibit the adherence ofcertain bacterial species to various substrates, is also highly enrichedin the content of flavonoids and polyphenols. The extract may be inpowder form or in solution in a suitable solvent. The powdered form is areddish-brown powder with a density of about 0.43±0.03 gm/cubiccentimeter and other properties as described in the subsequentparagraphs. For convenience and clarity, the extract will be referredhereinafter to as the "enriched extract", or, when reference is made toan extract made from a particular species such as cranberries, as the"cranberry extract".

Table I shows the solubility of one embodiment of a powder according tothe invention, in various solvents of differing degrees of polarity.

The refractive index of an aqueous solution of the powder is about1.3320 at a concentration of 1.0 mg/ml, and about 1.3370 at aconcentration of 8.5 mg/ml (the maximum solubility in water).

                  TABLE I                                                         ______________________________________                                               Solvent      Solubility                                                ______________________________________                                               Acetone      133 mg/ml                                                        Methanol     530 mglml                                                        Ethanol      450 mg/ml                                                        Butanol      275 mg/ml                                                        Water         8.5 mg/ml                                                ______________________________________                                    

The extract prepared from cranberries (berries of V. macrocarpon) hascertain characteristics observable in absorbance spectra in theinfrared, visible and ultraviolet light ranges. FIG. 2 shows aninfra-red transmittance spectrum of the extract, with significantabsorbance troughs at approximately the following respective wavenumbers in cm⁻¹ : 3410, 2960, 1735, 1610, 1524, 1443, 1360, 1285, 1210,1111, 1066, 822, 785, 500, which are respectively indicated by referencenumerals 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228. Peak 228 at about 500 cm⁻¹ is very broad.

FIG. 3 shows an absorption spectrum of the cranberry extract dissolvedin methanol, for wavelengths from about 250 nanometers (hereafterabbreviated "nm") through about 600 nm (the ultraviolet and visiblelight regions). The concentration of the extract in methanol is about0.05 mg/ml. Four major absorption peaks at wavelengths of about 202,278, 368, and 454 nanometers, are respectively indicated by arrows 310,320, 330, 340. These three peaks appear to be characteristic of theextract. The relative intensities of the peaks may be somewhat variable,but are generally in the range of about 1.8:0.64:0.12 respectively forthe peaks 310, 320, 330, 340. Polyphenols, includingflavonoid-containing compounds, are known to have UV/visible lightabsorbance spectra with similar features.

FIG. 4 depicts absorption spectra of the invented cranberry extractdissolved in water, at two different concentration levels (curves 400,402). Curve 402 is taken from a sample which is 200 times moreconcentrated than that of curve 400. The spectrum has the followingcharacteristic absorbance peaks: 202 nm, 278 nm, 368 nm, and 454 nm,which are indicated respectively by reference numerals 410, 420, 430 and440. The approximate relative intensities of the peaks are, in (mg/ml)⁻¹(cm)⁻¹, 42, 1.1, 0.3, and 3.6, respectively. Peaks Nos. 420, 430 (278 nmand 368 nm, respectively) are also characteristic of the spectra inwater of flavonoid- and polyphenol-containing compounds. Generally, anabsorbance at both 280 nm and 360-370 nm is indicative of the presenceof flavonoids, whereas polyphenols have the greatest absorbance in the200 nm to 280 nm range and exhibit little or no absorbance at 360-370nm.

The flavonoid/polyphenol compounds present in the extract includecompounds having a glycoside moiety. Several flavonoid compounds lackinga glycoside moiety, including myricetin, rutin, and quercetin, have beentested and found not to have anti-adhesion activity.

The cranberry extract is also characterized by the elution at specifictimes during high-pressure liquid chromatography (HPLC) of the extractdissolved in methanol, of 360 nm light-absorbing components. FIGS. 5Aand 6A depict typical elution vs. time chromatograms of the cranberryextract of components which absorb at 360 nm. Fifteen hundredmicroliters of a solution containing 100 milligrams of dry powder per 4milliliters of methanol (25 mg/ml; about 37.5 mg) was applied to apreparative-size reverse phase C18 column (Waters C18 prepacked column,BONDAPAK, cat. #WAT038505, Millipore Corp., Waters Chromatography, 34Maple St., Milford Mass. 01730). The C18 is a lipophilic affinity agenthaving carbon chains 18 carbons in length bound to the beads. For apreparative size column of about 50 ml volume, 25 mm diameter and 100 mmlength, a two-solvent linear gradient from 80% A/20% B (A=0.4%phosphoric acid, B=methanol) to 31% A/69% B is run at a flow rate of 5ml/minute over a time from 0 to 76 minutes. After 76 minutes, anisocratic flow at 31% A/69% B is maintained for an additional 44minutes. With photodetection at 360 nm, about four large 360 -nmabsorbing peaks are observed respectively at about 46 minutes, 52minutes, 56 minutes and 64 minutes (identified by reference numerals602, 604, 606, 608, respectively, in FIG. 6).

For an analytical column of about 5 ml volume (FIG. 8), about 1.25 mgpowder is loaded in a volume of about 50 μl. A two-solvent lineargradient from 80% A/20% B (A=0.4% phosphoric acid, B=methanol) to 31%A/69% B is run at a flow rate of 1 ml/min over a time from 0 to 38.3minutes. After 38.3 minutes, an isocratic flow at 31% A/69% B wasmaintained for an additional 21.7 minutes. With photodetection at 360nanometers, about four major elution peaks are observed at elution timesbetween about 22 minutes and about 40 minutes, and three smaller elutionpeaks appear earlier, at between about 9 minutes and about 15 minutes.

In some cases (FIG. 9), a 50 ml preparative column was first run forfive minutes at 80% A, followed by the linear gradient from 80% A to 31%A over the next 38.3 minutes, and finally by an isocratic phase at 31% Afor the succeeding 21.7 minutes (total time 65 minutes). With thisgradient protocol, the pattern of 360 nm-absorbing elution peaksassociated with the active fraction of the extract is somewhatdifferent, but closer to that observed for the analytical column.

The cranberry extract is also characterized by certain UV-fluorescingcomponents migrating in a specific pattern in paper and thin-layerchromatography (TLC). When a solution of the cranberry extract issubjected to TLC on silica gel in a 98:2 acetone:water solvent, fourcharacteristic fluorescing bands may be observed under long-waveultraviolet light. These characteristic bands have the properties asdescribed in Table II:

                  TABLE II                                                        ______________________________________                                        Relative migration distance (R.sub.f) and color upon irradiation              with ultraviolet light for characteristic bands observed                      by thin-layer chromatography in acetone:water (98:2).                                  Rf          Color                                                    ______________________________________                                        Band #0    0.0           faint red                                            Band #1    0.2           blue/blue-white                                      Band #2    0.6           bright yellow                                        Band #3    0.7           bright yellow                                        Band #4     0.97         white                                                ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        Relative migration distance (R.sub.f) and color of fluorescent                bands observed upon irradiation with long-wave ultraviolet                    light, for paper chromatography in 6:1:2                                      butanol:acetic acid:water.                                                            Rf       Description                                                  ______________________________________                                        Band #5   0.44       Strong fluorescent yellow                                Band #6   0.50       faint fluorescent yellow                                 Band #7   0.77       medium fluorescent yellow                                ______________________________________                                    

When subjected to paper chromatography on a Whatman #3 paper with asolvent system of 6:1:2 butanol:acetic acid: water, three significantfluorescent peaks are observed on exposure to long-wave (366 nm) UVlight. These three peaks are described in Table III.

In a preferred embodiment, the extract (whether from cranberries orother Vaccinium species) is substantially free of free simple sugars(monomer and dimer sugars such as fructose, galactose, glucose, sucrose,etc).

In a further embodiment, the total acid content of the extract(including benzoic acid) is less than about 2%. Benzoic acid is usuallypresent at less than about 0.005 mg per gram. This benzoic acid contentis ≦ about 1% of that found in certain existing products, as summarizedin Table III. Juice products were reduced to a powder before testing andthe acid content is given in relation to the weight of the resultingsolids.

                  TABLE IV                                                        ______________________________________                                        Product             Wt. % Benzoic Acid                                        ______________________________________                                        Knudsen cranberry juice                                                                           0.50                                                      Hains cranberry juice                                                                             0.11                                                      Janet Lee cranberry juice                                                                         0.06                                                      Ocean Spray cranberry cocktail                                                                    0.10                                                      Ocean Spray cranberry powder                                                                      0.12                                                      Cranberry extract of application                                                                  0.0002                                                    ______________________________________                                    

The reduced content of benzoic acid and total acid results in a productwith a less sour taste and which is less likely to cause stomach upsetor promote tooth decay.

The cranberry extract having the foregoing properties including a verylow content of simple sugars and benzoic acid, has been found tointerfere with adherence of bacterial cells to certain cell types, aswell as to surfaces such as polystyrene. Examples 1-3 following,demonstrate the anti-adherence properties of the extract.

At least three modes of bacterial cell adherence to other cells andsurfaces are known. One mode is mediated by type 1 pili on the surfaceof the bacteria, and is characterized by sensitivity to free mannose. Asecond mode is mediated by P-type pili. The mechanisms of the third modeand other adherence modes, are not well-characterized. Guinea pig RBCsare believed to have receptors for the type 1 (mannose-sensitive) piliof E. coli, since the bacteria are capable of agglutinating guinea pigRBCs in the absence of mannose but not in its presence (see for exampleAronsen, J. Infect. Dis. 139:329-332, 1979; Riegman, J. Bacter.172:1114-1120, 1990; Jann, Infect. Immun. 22:247-254, 1981).

EXAMPLE #1

Anti-adherence activity measured as interference with bacterialadherence to bladder cells:

Human bladder epithelial cells are collected by centrifugation from theurine of a healthy female volunteer. The cells are washed in standardsaline citrate (SSC) and resuspended to the desired volume. The opticaldensity of the cell solution and cell counts are determined. E. colibacterial strains isolated from urinary tract infections are cultured intryptic soy broth at 37° C. for 72 hours to encourage piliation. Thebacteria are also harvested by centrifugation, resuspended in thedesired volume of SSC, and the approximate cell number determined.

A test tube is prepared containing 0.667 ml of a selected dilution ofthe substance to be tested for anti-adherence activity, plus 0.334 ml ofthe bacterial suspension. The bacteria are incubated with the testsubstance for 15 minutes at 37° C. Next, 1.0 ml of the bladder cellsuspension is added to each tube and the tubes are incubated for afurther 15 minutes. Cranberry extract was used in an 8.5 mg/ml solution.Unfractionated cranberry juice was also tested.

After incubation, the content of each tube is filtered through an 8micron polycarbonate filter, and the filter is rinsed with two volumes(2 ml=one volume) of SSC to wash free any bacteria which are not adheredto the bladder cells. The filter is placed face down on a microscope andthe cells are heat-fixed to the slide. The filter is removed, the slidesare stained to visualize the cells, and the number of bacteria adheringper cell is counted for each of 20 cells per slide.

Two control tests are also performed. One, the bladder cells areincubated only in SSC without any bacterial suspension or testsubstance, to determine how many bacteria originating in the urinesample are attached to the epithelial cells. Second, SSC is substitutedfor the test substance to determine the maximum number of bacteria thatadhere to the cells without any inhibitor.

EXAMPLE #2

The following test was developed and used to test the ability of theextract to inhibit agglutination of guinea pig RBCs by E. coil:

Red blood cells (abbreviated RBCs) from guinea pigs, purchased fromMicrobio Products (Tempe, Ariz.) as a suspension in Alsevers solution,are washed in SSC (standard sodium citrate, as known in the art) andresuspended in SSC. Human red blood cells are obtained by standardmethods from the blood of volunteers and also prepared by washing andresuspending in SSC. Human RBCs are believed to have a receptor formannose-resistant pili of E. coil.

E. coil cultured and prepared as described in the previous example aretested as follows. A series of dots containing graded amounts of thetest substance diluted in SSC, plus one dot containing SSC only, areplaced on a polystyrene plate. The dots may have a volume of about 10μl. An equal volume of bacterial suspension is mixed into each dot,followed by 1/2 volume of RBCs. The total volume in each dot, for astarting volume of 10 μl per dot, is thus 25 μl. The contents of eachdot are thoroughly mixed and the degree of agglutination is scored on ascale of 0-4, with 0 representing no agglutination. The sum of the dotsat different dilutions is totalled and subtracted from 32 to provide anActivity Index of activity interfering with agglutination.

FIGS. 5A, 5B and 6A, 6B depict absorbance chromatograms fromhigh-pressure liquid chromatography (preparative-type column, 25millimeters by 100 millimeters). In FIG. 5A, absorbance at fourdifferent wavelengths is shown: 230 nanometers (abbrev. nm), 280 nm, 360nm, and 512 nm. Flavonoids and polyphenols are known to have significantabsorbance at about 280 nm, with flavonoids also having an absorbancepeak at about 360 nm; anthocyanins are known to have significantabsorbance of 512 nm light. FIG. 5B is a magnified duplicate of thechromatogram at 512 nm of FIG. 5A. From a comparison of FIGS. 5A and 5B,it can be seen that the peak heights of peaks corresponding toanthocyanin absorbance are about 1/10 or below, the peak heights ofpeaks corresponding to polyphenol absorbance (280 nm). From the relativepeak heights it can be seen that the amounts of anthocyanins in theextract are less than 10% of the amounts of polyphenols. In fact, as theanthocyanin extinction coefficients are much higher than those ofpolyphenols, the amount of anthocyanins by weight is believed toactually be much lower, possibly as low as 1%.

Twelve fractions 502, 504, 506, 508,510, 512, 514, 516, 518, 520, 522,524 were separately collected and further analyzed. Fractions 510, 514,518, 522 contain significant peaks absorbing at about 350 to about 370nanometers, wavelengths characteristic of flavonoid and polyphenolmoieties. Fractions 504 and 506 span the retention time of anthocyaninpeaks absorbing at 512 nm.

Table V compares the anti-adherence activity of the fraction 502 through524 with that of a whole (unfractionated) extract. A chromatogram of thelatter sample is depicted in FIG. 9 (obtained by the alternativegradient procedure with 50 ml size column). Charts of the absorbancespectrum of fractions 510, 514, 518, 522, 504, and 506 are depicted inFIGS. 7A-7F, respectively.

From these spectra, it is apparent that fractions 510, 514, 518 and 522all contained relatively large quantities material absorbing at about350-360 nm. Additionally, as seen in Table V, substantial anti-adhesionactivity was found in fractions 504, 506 which did not containdetectable amounts of 360 nm-absorbing material. Fractions 504, 506contain small amounts of compounds absorbing at 512 nm (which arebelieved to be anthocyanins), and substantial amounts of materialabsorbing at 230 to 280 nm. However, a cranberry anthocyanin preparationwas analyzed and found not to contain detectable anti-adhesion activity(see Table VI). Thus, it appears that at least some of the cranberryanti-adhesion activity is found in some compounds absorbing at 230 to280 nm.

The strain of E. coli used for the tests on adherence to human bladder

                  TABLE V                                                         ______________________________________                                        Comparison of HPLC Fractions in Guinea Pig RBC                                Agglutination Assay                                                           Fraction #      Activity Index                                                ______________________________________                                        510             6                                                             512             4                                                             514             5                                                             516             2                                                             518             1                                                             520             0                                                             522             0                                                             524             0                                                             526             4                                                             504             18                                                            506             18                                                            508             6                                                             500             0                                                             502             5                                                             Step 116 Extract                                                                              9                                                             ______________________________________                                    

cells was isolated from an active bladder infection in a human subject.This strain, designated the #3B strain, appears to possess both type 1and P-type pili (the latter are sometimes referred to in the researchliterature as "P-type fimbriae").

Results of the agglutination test for various substances and for the twoE. coli strains are shown in TABLE VI. Gp indicates guinea pig cellassay; HU indicates human cell assay. In the guinea pig assay, thehighest concentration of final extract (step 116) in a 25 μl test dotwas 0.056 mg/25 μl; the highest amount of anthocyanins in a dot was0.063 mg/25 μl; the highest amount of mannose was 0.10 mg/25 μl.

                  TABLE VI                                                        ______________________________________                                        Comparison of Adhesion Inhibition by Extract                                  to that by Known Substances                                                   Sample ID       Blood   Activity Index                                        ______________________________________                                        1% mannose      Gp      24                                                    Final Extract   Gp      10                                                    EXPT. 1         Hu      13                                                    Final Extract   Gp       9                                                    EXPT. 2         Hu      11                                                    Alcohol Extract Gp      10                                                    Anthocyanins    Hu       0                                                    Anthocyanins    Gp       0                                                    ______________________________________                                    

Results of a similar test performed on a sample of the acidified alcohol(step 102) extract, with the maximum amount being 0.4 mg/25 μl dot, arealso shown. The anthocyanins used in the experiment shown in Table VIwere obtained from cranberries by a procedure described subsequentlyherein and outlined in FIG. 10. In Step 1008B, the elution of the cationcolumn with 1% HCl, after collection of the void volume and aqueouswashes (step 1008A), is found to selectively recover much or all of theanthocyanin content of cranberrry. The anthocyanin preparation did notcontain significant amounts of other substances.

From the results in Tables V and VI, it is apparent that the cranberryextract inhibits both type 1 pili-mediated adhesion of E. coli to guineapig RBCs, and adhesion mediated by P-type pill. Since the extractcontains virtually no free monomer or dimer sugars, the inhibition ofP-type adhesion cannot be attributed to such sugars. Interestingly,P-type adhesion is believed to occur at high levels in E. coli inurinary tract infections.

The extract also reduced the adherence of Pseudomonas aeruginosa tobladder epithelial cells, although to a lesser degree than observed withE. coli. There was no apparent effect upon the adherence of severalLactobacillus strains to human bladder cells.

Additionally, E. coli did not adhere to polystyrene plastic in thepresence of the extract. However, it should be noted that in general E.coli do not tend greatly to adhere to polystyrene.

An extract having the characteristics of the enriched extract describedhereinabove may be prepared by the steps illustrated in FIG. 1. Exceptfor the final non-polar solvent extraction, which selectively removesanthocyanins, this method is similar to a standard method useful toextract anthocyanins from plant materials (see for example OfficialMethods Of Analysis of the Association of Official Analytical Chemists,sections 22.092 through 22.095 et seq., pages 424-425, 1984; also"Purification of Cranberry Anthocyanins", by T. Fuleki and F. J.Francis, J. Food Science 33:266-274, 1966.) Anthocyanins are flavonoidcompounds closely related to, and often co-isolating with, polyphenolcompounds. However, cranberry-derived anthocyanins tested by the methodsdescribed hereinabove did not significantly inhibit bacterial adhesionto surfaces. Moreover, cranberry extract which is about 1000-foldenriched for the adhesion-inhibiting activity, has low or no levels ofanthocyanins. Anthocyanins characteristically exhibit strong absorptionof 512 nanometer light. From the chromatograms of FIGS. 5A and 5B, it isapparent that the levels of anthocyanins in the extract were at leastabout tenfold lower than the levels of polyphenol components absorbingat about 360 nanometers.

The method depicted in FIG. 1 also removes substantially all the simplesugars and most of the benzoic acid from the enriched extract. However,other methods are known for concentration and purification ofpolyphenols and related compounds such as flavonoids, anthocyanins andcatechols from cranberries and other plant materials (see for example G.Puski and F. J. Francis, "Flavonol glycosides in cranberries", J. FoodScience 32:527-530, 1967; T. Fuleki and F. J. Francis, "Quantitativemethods for anthocyanins: purification of cranberry anthocyanins", J.Food Science 33:266-274, 1968; P. L. Wang et al., "Isolation andcharacterization of polyphenolic compounds in cranberries ", J. FoodScience 43: 1402-1404, 1978; the contents of which are herebyincorporated by reference). Such other methods may also produce anextract enriched for anti-adhesion activity, but having greater orlesser amounts of peripheral substances such as the previously mentionedsugars, benzoic acid, and anthocyanins.

A method for preparing an enriched extract is as follows. First, polarand charged compounds including anthocyanins and other flavonoid andpolyphenol compounds are extracted from plant material of plants of thegenus Vaccinium (step 100). Preferably, the plant material is berries orfruits. A presently preferred embodiment uses cranberries (V.macrocarpon).

In the preferred embodiment, step 100 involves crushing wholecranberries, mixing them with a large volume of acidifed alcohol, andthoroughly agitating the mixture. The acidified alcohol preferablycomprises a fairly polar alcohol and water in about equal proportions,with a suitable acid in an amount of about 3 to about 10% by volume. Inthe presently preferred embodiment, the proportions are 10:1:10ethanol:acetic acid:water. Suitable alcohols include methanol, ethanol,and propanol, and suitable acids include acetic acid, hydrochloric acid,and phosphoric acid.

Step 100 produces a liquid extract containing mainly polar and/orcharged compounds including the active fraction, with a solid residue ofberry debris containing largely nonpolar compounds. The solid residue isdiscarded.

The liquid extract is then concentrated to about 4-6% of the originalvolume of extract (step 102), with removal of a substantial part of thealcohol. Typically, the extract is evaporated to perhaps about 1-5% ofthe original volume of liquid extract, and then water is added to makeit up to 4-6% of the original volume. The result is a concentratedliquid extract.

Next (step 104), monomer and dimer sugars are substantially removed fromthe concentrated extract. In the working embodiment, a metal acetate orsulfate, preferably zinc or magnesium acetate or zinc sulfate, is addedto the concentrated liquid extract to cause formation of a solidprecipitate leaving the simple sugars in solution. The precipitateincludes complexes of the active fraction with the metal. Preferably,step 104 is carded out by adding sufficient metal compound to theextract to a concentration of about 1 molar to 1.1 molar, followed byabout 0.4 volumes of a volatile inorganic base are added to theconcentrated extract and thoroughly mixed. The inorganic base isdesirably rather strong; in the illustrated embodiment, ammoniumhydroxide is used. Formation of a solid precipitate occurs relativelyrapidly upon addition of the base.

Of the metal salts tried, lead acetate produces the greatest enrichmentfor the active fraction. However, lead acetate may be undesirabledepending upon the final use of the product active fraction. Forexample, trace amounts of lead in a culture vat cleaned with the activefraction might have deleterious effects on subsequent cultures, or endup in food products produced from the cultures. Lead acetate would alsobe undesirable if the active fraction were to be used in orally ingestedcompositions, for example a mouthwash for inhibiting bacterial adhesionto teeth, or a health product.

Where the use of a lead compound is undesirable, other metal salts maybe used, for example zinc, magnesium, nickelous, barium and calcium,cobalt or sodium acetates, or zinc sulfate. Of these, zinc acetate,magnesium acetate, and zinc sulfate are found to produce the greatestenrichment for the active fraction. Nickelous, barium and calciumacetates produced an enrichment of 60-80% relative to that achieved withzinc acetate (taken as 100%), while sodium acetate and cobalt acetategave relative yields of only 20-40%.

Desirably, the precipitated solids are washed with a large amount of apolar alcohol such as 80% ethanol in water, to remove excess salt andtrace remnants of monomer and dimer sugars (step 106). Methanol orpropanol may be substituted for ethanol.

The washed solids are then mixed with n-butanol and concentratedhydrochloric acid, in a proportion of about 6:1 (step 108). Then-butanol removes the metal ion and solubilizes the active fraction,while the hydrochloric acid is believed to alter the relative polarityof the active components to thereby render them soluble in n-butanol.Although n-butanol is presently preferred, other moderately polarsolvents such as i-butanol, t-butanol, pentanol or hexanol may besubstituted for n-butanol. The result is an alcohol liquid phase and apellet comprising mostly metal chloride; the pellet is discarded (step109).

The butanol is then removed from the liquid phase from step 108 byextraction with a nonpolar organic solvent such as petroleum ether (step110). About three to about ten volumes of the organic solvent are addedto the alcohol liquid phase and vigorously mixed, then let stand toallow separation into a hydrophobic first organic phase comprisingpetroleum ether and butanol, and a first aqueous phase which containsthe active fraction. The first aqueous phase, which is generallyorange-red to red-brown in color (due at least in part to the presenceof anthocyanins), is separated from the organic layer and diluted withabout 4 volumes of water (step 112).

Highly desirably, the first organic phase is back-extracted with water(1/20 volume) until all of the red color has been removed from the firstorganic phase. The water layers from the back-extractions are thencombined with the aqueous phase of step 110, before the dilution of step112 and subsequent steps.

After dilution, the first aqueous phase is then further extracted with amoderately polar organic such as ethyl acetate (step 114) to produce asecond organic phase containing the active fraction, and a secondaqueous phase in which a substantial proportion of the anthocyaninsremains. Preferably, the first aqueous phase is extracted three timessequentially with about an equal volume of ethyl acetate, and the threeethyl acetate phases are pooled. Alternatively, the extraction step 114may be performed as two sequential extractions, the first with an equalvolume of diethyl ether, the second with an equal volume of ethylacetate.

In either case, following extraction 114 the second organic phase isseparated from the second aqueous phase and evaporated to leave a pasteor powder which is generally orange in color and having a density ofabout 0.430 g/cm³. The powder has a solubility in water of about 8.5grams per milliliter. This product will for convenience be termed a"flavonoid-enriched extract", which is consistent with the spectral dataindicating enrichment for flavonoid-containing compounds. However, theterm is not intended as limiting the active fraction of the extract toflavonoids or the like. The anthocyanins are substantially selectivelyretained in the second aqueous phase of extraction 114, although somemay remain in the second organic phase. However, the second aqueousphase has considerably less anti-adhesion activity than the secondorganic phase, indicating that the active fraction probably does notinclude significant amounts of anthocyanin compounds.

The extract prepared by the process described in the precedingparagraphs is enriched about 1000-fold for an active fraction whichinhibits the adhesion of bacteria, including E. coli and Pseudomonasaeruginosa, to mammalian cells and to certain surfaces. There is nodetectable protein, and little or no free monomer and dimer sugars, inthe enriched extract. Those free simple sugars remaining are primarilysimple reducing sugars such as glucose and fructose. The caloric valueof the extract is generally less than about 4 calories per gram. Also,the acid content of the enriched extract, especially the benzoic acidcontent, is considerably lower than that of Vaccinium berries and ofmany other cranberry extract or powder products presently known.

EXAMPLE #3

Comparison of extract yields using different metal compounds in Step104:

Four hundred twenty (420) grams of cranberries were ground with 420 mlof acidified alcohol, and the mixture was let to stand overnight. Themixture was centrifuged, the supernatant #1 separated and set aside, andthe solid pellet #1 ground with an additional 1200 ml of acidifiedalcohol. The ground pellet #1 mixture was centrifuged, the supernatant#2 set aside, and the pellet #2 was in turn ground with 1000 milliliters(abbreviated hereinafter as "ml") of acidified alcohol. The groundpellet mixture #2 was again centrifuged and the supernatant #3 wascombined with supernatants #1 and #2.

The combined supernatants were evaporated to a volume of 450 ml having apH of about 1.7. The mixture was divided into six portions of about 75ml each. To each of the 75 ml portions, 20 ml of 15 molar ammoniumhydroxide was added, bringing the solution to a pH of about 8.5 to 8.8.Individual samples were then respectively mixed with 100 ml of a 1.1molar solution of one of the following: zinc acetate, zinc sulfate,calcium acetate, barium acetate, cupric acetate, cobalt acetate. Thesamples were centrifuged, the supernatant discarded, and each pellet waswashed three times with 100 ml of 80% ethanol in water each time. Thewash supernatants were also discarded.

Next, to each pellet 20 ml of n-butanol and an amount of concentratedhydrochloric acid sufficient to reduce the pH to less than about pH 2.5was added (step 110; generally about 0.75 ml of HCl with certainexceptions as noted in Table VII. The zinc sulfate-treated sampleproduced a relatively large pellet to which it was necessary to add anadditional 0.8 ml of HCl to achieve satisfactory solubilization of thepellet. For the barium acetate sample, an additional 0.25 ml of HCl wasneeded to react the pellet. The pH of all the samples ranged betweenabout 0.8 and 2.5.

The six samples were again centrifuged and the pellets discarded. Thebutanol supernatant of each of the six samples was subjected topetroleum ether extraction (step 112), with a sufficient number ofback-extractions with water to transfer essentially all the red colorfrom the organic phase to the aqueous phase. Results of the petroleumether extraction for the six samples (step 112) are summarized in TableVII.

The aqueous layer of each sample was then extracted 3 times with 50 mlethyl acetate (Step 114). The zinc acetate-treated sample yielded about150 ml of orange-red ethyl acetate phase. The zinc sulfate sampleyielded about 150 ml of dark red solution. The calcium acetate sampleproduced about 150 ml of a yellow-orange solution. The bariumacetate-treated sample produced about 150 ml of a light yellow solution,while the cupric acetate sample yielded about 150 ml of a intense yellowsolution, and the cobalt-treated sample resulted in about 150 ml of ayellow solution.

The separated ethyl acetate phase of each of the samples were evaporatedto dryness (except for the zinc sulfate sample, which did not becomefully dry), producing a red powdery residue. The recovered weight of thedry extracts was as follows: zinc acetate, 63 mg; zinc sulfate, 2980 mg;calcium acetate, 31 mg; barium acetate, 113 mg; cuptic acetate, 14 mg;cobalt acetate, 26 mg.

                  TABLE VII                                                       ______________________________________                                                    Vol. Petr. No.        Aqu. End                                    Sample      Ether      Back-Extr. Vol.                                        ______________________________________                                        EXAMPLE #3                                                                    Zinc acetate                                                                              100 ml     2          30 ml                                       Zinc sulfate                                                                              300 ml     1          25 ml                                       Calcium acetate                                                                           100 ml     2          30 ml                                       Barium acetate                                                                            300 ml     3          50 ml                                       Cupric acetate                                                                            100 ml     2          30 ml                                       Cobalt acetate                                                                            100 ml     2          30 ml                                       EXAMPLE #4                                                                    Zinc acetate                                                                              100 ml     3          40 ml                                       Nickelous acetate                                                                         100 ml     3          30 ml                                       Magnesium acetate                                                                         100 ml     3          30 ml                                       Sodium acetate                                                                            100 ml     3          25 ml                                       Ammonium acetate                                                                          100 ml     3          20 ml                                       ______________________________________                                    

EXAMPLE #4

A second comparison of yield using different metal compounds in Step 104was performed in the same manner as for Example #3. The results areincluded in Table VII. The dry weight yields were: zinc acetate, 46 mg;nickelous acetate, 14 mg; magnesium acetate, 17 mg; sodium acetate, 18mg; ammonium acetate, 1 mg.

FIG. 8 shows a chromatogram of the initial liquid extract resulting fromstep 102, made from cranberries. The liquid extract (the aqueoussupernatant after removal of the alcohol) had a volume approximately thesame as the starting volume of cranberries (see Example III). Thechromatogram of FIG. 8 differs from that of FIGS. 5A, 5B, 6A, and 6B inthat a different column size, with 4 μm particle size packing, was usedwith a slightly different procedure. To obtain the chromatogram of FIG.8, an analytical C18 column of dimensions 8 millimeters diameter, 100millimeters length and a volume of 5 ml was used. The concentration byweight of material in the extract was about 200 mg per ml (determinedfrom an aliquot of the liquid extract evaporated to dryness). Fiftymicroliters (abbrev. μl) of a 200 mg per ml solution (total mass 10 mg)was injected into the column and a flow rate of 1 ml/min was used. Underthese conditions, the most prominent elution peaks 802, 804, 806absorbing at 360 nanometers have retention times of about 33 minutes, 36minutes and 38 minutes, respectively.

FIG. 9 depicts a chromatogram of the cranberry extract resulting fromstep 116, eluted by the alternate protocol on a preparative-size (50 ml)C18 column. In this case, 50 μl of a 50 mg per ml solution of thecranberry extract, was injected onto the column (2.5 mg total mass).Three prominent 360 nm-absorbing peaks 902, 904, 906 are also seen inthe chromatogram of FIG. 9, having retention times of about 34 minutes,39 minutes, and 43 minutes.¹ These peaks are slightly shifted from thoseof FIG. 8, by an amount consistent with the 5-minute difference intiming of the gradient between the analytical column of FIG. 8 and thealternate (shortened) preparative column protocol.

The following computation provides a rough estimate of the relativeenrichment for 360-nm absorbing compounds (flavonoids) in the finalcranberry extract. The area under the peaks absorbing at 360 nm in FIG.9 is about 2.6 A.U. (absorbance units), while that for the 360m-absorbing peaks in FIG. 8 is about 0.12 A.U. Dividing 2.6 by 0.12, andmultiplying the result by 4 the factor of the difference in mass appliedto the column (4 times as much material loaded for FIG. 8 as for FIG.9), the enrichment for 360-nm absorbing compounds in the cranberryextract of step 116 over the levels in the initial liquid extract, isseen to be about 87-fold.

From the rough computation and similar rough estimates made from othersamples, it appears that the degree of enrichment for 360-nm absorbingcompounds (which are presently believed to be polyphenols), is similarto the degree of enrichment for anti-adherence activity in the finalcranberry extract.

Further preferred modifications and alternate embodiments of the processand the resulting extract are as follows.

In the process depicted in FIG. 1, it has been found that in the step ofethyl acetate extraction (step 114), a significantly higher yield bothin terms of mass and in terms of activity level per unit mass isobtained if the extraction is performed under acidic conditions.Desirably, the extract at this stage has a pH of about 2 or less. Ifneeded, acidification is accomplished by addition of HCl.

A variety of methods may be used to obtain an initial aqueous Vacciniumextract from which the anti-adhesion activity may be obtained. TableVIII includes a comparison of activity obtained using a selection ofextraction methods.

Also, it has been found that in lieu of fresh or frozen berries, anotheruseful starting material is an aqueous solution of a powdered cranberryproduct (commercially available under OCEAN SPRAY brand). This solutionmay be used in place of the acidified alcohol-water extract of wholeberries (steps 100-102 of FIG. 1). The cranberry powder is believed tobe obtained by spray-drying of an aqueous extract of the raw cranberrymaterial. A solution of 10% by weight of powdered cranberry is astarting material that has yielded good results. Higher concentrationsof this powder may leave some material not fully dissolved. In aprocedure starting with such a cranberry solution, the steps 100, 102 ofextraction of whole berries and removal of alcohol from the extract areeliminated.

An alternate embodiment (FIG. 10) involves processing the initialaqueous extract largely by chromato-graphic separation steps instead ofby precipitations, phase separations etc. This embodiment is presentlypreferred for convenience and because it reduces the use of organicsolvents. However, the chromatographic embodiment is still directed tothe fundamental steps of enriching for charged and polar compounds,removing monomer and dimer sugars, removing benzoic acid and otherorganic acids, and removing anthocyanins, and these steps could beaccomplished by substituting the appropriate steps previously describedin reference to FIG. 1. That is, a "hybrid" embodiment combining certainsteps from the embodiment of FIG. 1 and some from the embodiment of FIG.10, may also produce the desired extract.

For example, in the embodiment of FIG. 10, the removal of simple sugarsand benzoic acid from the extract (steps 104, 106, 108-111) isaccomplished by reverse-phase affinity chromatography on a lipophiliccolumn, such as the C18 also used for HPLC analysis of the extract (step1000).

The chromatographic embodiment may desirably include a further step ofselectively separating compounds which compete with mannose for bindingto a mannose-affinic substrate. This step is accomplished by subjectingthe extract to affinity chromatography on a mannose-binding substratebound to a support. In the present working examples, a concanavalin-A(abbreviated conA) column which is commercially available from SigmaChemical Co. is used. ConA is known to bind mannose, and mannose bindsto type 1 pili and interferes thereby with the pili-mediated adhesion oftype 1 pili. In the conA affinity chromatographic step, the extract ispassed over the column to cause selected components to bind to the conA,and non-conA-binding compounds are washed from the column. A solutioncontaining a sufficient excess of mannose or a mannose derivative isthen used to elute the selected components from the column bycompetition.

It will be apparent that other mannose-binding agents could be similarlyused for a mannose affinity chromatography separation. An alternateembodiment presently contemplated involves isolation of the pili and/orthe mannose binding region of the pili, and use of this in place of conAfor affinity chromatography. However, conA-conjugated supports arepresently commercially available and thus convenient.

Highly desirably, the mannose affinity chromatography is performed afterthe extract has been partially purified by other steps. For example, theextract should already be free of simple sugars, which could bind toconA and thus interfere with the binding of the desired polyphenol andflavonoid compounds.

A presently preferred embodiment of the method for chromatographicpreparation of the extract is depicted in FIG. 10. The first step 1000of the procedure is to remove the monomer and dimer sugars and benzoicacid from a primary aqueous Vaccinium extract, by subjecting the primaryextract to reverse-phase liquid chromatography on a lipophilic column.The primary extract can be obtained by making an aqueous solution of apowdered starting material as described above, by making an acidifiedalcohol extract as by steps 100-102 of the method of FIG. 1, or by otherequivalent means.

In a working embodiment, the reverse-phase chromatography of step 1000is performed on a C18 column (Waters C18 "BondaPak", 10 μm beads,available from Millipore Corp., Waters Chromatography, 34 Maple St.,Milford Mass. 01730, cat. #WAT038505). A 50-ml C18 column is preparedessentially according to the manufacturer's directions, by flushingfirst with 100 ml methanol and then 200 ml distilled water at a flowrate of 4-6 ml/min. From 350 ml to about 500 ml of the aqueous primaryextract is then loaded onto the C18 column at a flow rate of 2.0ml/minute to about 0.5 ml/minute. If the fluid leaving the column duringloading becomes pink or red in color, this indicates that the column isbeing saturated. The column is then washed with an excess of water(800-1000 ml, or a sufficient amount until the eluant appears colorlessor hint pink), at a flow rate of about 4-6 ml/min, to remove the sugars.Next, a sufficient volume of methanol, typically about 150 ml to about300 ml, is run through the column to elute the desired compounds. Themethanol eluate should be a deep red; when the eluate becomes pale pinkthen sufficient methanol has been passed through the column. Themethanol eluate usually contains precipitate which is readily removed bycentrifugation at 1500 to 5000 rpm with a tabletop or slightly largercentrifuge (step 1004). The precipitate is discarded and the desiredactivity is found in the supernatant.

The methanol supernatant of step 1002 is then evaporated under vacuum toevaporate all of the methanol, and redissolved in about 25 ml warmwater, preferably with sonication (step 1004). The sample is filteredwith a Whatman #1 filter to remove precipitate, which is discarded.

The aqueous sample is then subjected to chromatographic separation on acation column (step 1008). A 4 ml to 8 ml cation column is prepared asgenerally known, from a slurry of cation material in water. A presentlypreferred cation material is the Waters Accell+CM Cation, which is asilica bead with a hydrophilic bonded layer having carboxymethyl as theavailable cation group (Millipore Corp., Waters Chromatography, 34 MapleSt., Milford Mass. 01730). In a typical procedure, the aqueous samplewhich has a volume of about 5 ml is carefully loaded directly onto thecation column. At this point, a green color will be observed in thecolumn and a very dark green material at the top of the column. Thecolumn is then washed with about 25 ml of distilled water (step 1008A).The dark green material at the very top washes out only very slowly withplain water, and is desirably left behind.

The initial loading solution and the water wash (the void volume; step1008A) of the cation column contain a substantial amount of the desiredanti-adhesion activity. A substantial portion of anthocyanins areremoved from the extract by the cation column, as revealed by analyticC18 chromatography of products 1006 and 1008A (also subjected to theoptional CHCl₃ /EtAc extraction). Desirably, the column is now washedwith 1-2 volumes of 1% hydrochloric acid (step 1008B), which elutesessentially all the green material. The HCl eluate has been found toconsist mostly of anthocyanins. Following the HCl elution, a step ofmethanol elution (step 1008C) is then performed. The methanol eluate1008C, upon evaporation and resuspension in water, is surprisingly foundto contain a substantial amount of anti-adhesion activity. The methanoleluate compound(s) have significant HPLC elution peaks observable byabsorbance at 230 nm, 280 nm and 360 nm.

The void volume eluate 1008C is then concentrated by evaporation to aconcentration of less than about 60 mg/ml, in preparation for affinitychromatography on a concanavalin A column (conA). A 3 ml to 10 ml columnof conA-bound column material (commercially available from Sigma, St.Louis Mo., cat. #C-9017) is prepared and flushed with 100 ml ofphosphate buffer (0.05M sodium phosphate, adjusted to pH 7 withphosphoric acid) at a flow rate of about 1 ml/minute. Two ml of theaqueous eluate, containing no more than 60 mg/ml sample, is adjusted tothe same concentration of conA phosphate buffer and applied to thecolumn. After loading, the column is washed with about 50 ml ofphosphate buffer or until the eluate is clear, again at about 1ml/minute. Finally, the column is eluted with at least 50-100 ml ofphosphate buffer containing 10% α-methylmannopyranoside. Themannopyranoside compound is preferred over mannose for this purposebecause it binds more tightly to the conA, and thus is expected to moreeffectively elute compounds bound to the mannose-affinic site onconcanavalin A. A 10% concentration of α-methyl-mannopyranoside is foundto be more efficient at eluting the active fraction, than a lowerconcentration of 2%.

The resulting conA-eluate is then subjected to another separation on aC18 column similar to step 1000, to remove the α-methyl mannopyranoside.However, a smaller C18 column (Waters C₁₈ Sep-Pak, 1 ml volume,Millipore Corp., Waters Chromatography Division, 34 Maple St., MiffordMass.; cat. #WAT051910) is sufficient with the more purified sample atthis stage. This step also serves to concentrate the active fraction.The column is prepared by passing 5-10 ml methanol followed by 5-10 mlwater over it before loading with the 50 ml of conA eluate. The columnis then washed with 10-20 ml water, and the active fraction is eluted ina volume of about 2 ml methanol. The sample may be evaporated to drynessand resuspended in a volume of about 150 μl of distilled water.

The resulting extract is pale yellow to brown, indicating that itcontains little or no anthocyanins. A large part of the anthocyanins arelost in the cation column separation.

Optionally, a step of extracting the aqueous sample with an equal volumeof a moderately polar organic solvent, preferably 50:50 CHCl₃ /ethylacetate, may be introduced into the procedure of FIG. 10. This step isperformed to remove some nonpolar or less polar compounds from theextract. Chloroform-EtAc extraction may be performed either after step1004, or prior to steps 1000, 1002, e.g. on the initial aqueous extract.

The chloroform-EtAc extraction appears to remove a component of theextract which masks or otherwise interferes with the activity of theanti-adhesion fraction in the RBC assay, since its removal generallyenhances the activity as measured in that assay without significantlychanging the mass yield. However, removal of the apparent "masking"component(s) may decrease the long-term stability of the activecompound. Thus, the step of chloroform/ethyl acetate extraction shouldbe employed when attempting to quantitate anti-adhesion activity in theRBC assay, but it may be undesirable to include this step in routineproduction of the extract.

In performing the chloroform/ethyl acetate extraction, the aqueoussample is preferably extracted twice (each time with an equal volume ofchloroform/ethyl acetate). The organic phase(s) are discarded, and theaqueous phase is concentrated by evaporation to about 1/5 its originalvolume (e.g., around 5 ml). The evaporation also removes trace amountsof the organic solvents. Alternatively, the sample can be evaporated todryness and resuspended in water. Either way, the resulting aqueoussample is then further purified by step 1008 or step 1004.

Table VIII shows the relative adhesion-inhibiting activity measured forextracts at selected stages in the process of FIGS. 1 and 10, asmeasured in the red blood cell agglutination assay (abbreviated RBCassay; see Example 2 and Table VI) is given. The activity values arenormalized to the activity of a 1% mannose solution in the assay and tothe amount of material in grams per milliliter. The mannose solution is1% or 1 g/100 ml, so a value of 100 indicates activity equivalent tomannose. Values>100 indicate that the sample is more effective thanmannose in anti-adhesion activity. The concentrations of the testextracts were determined by weighing portions evaporated to dryness.

The results shown in Table VIII demonstrate that in general differentmethods of producing the initial extract were comparable, with the MAW,water (room temperature) and boiling water extracts being slightlybetter in terms of recovery of activity. The 10% solution of Ocean Spraypowder was comparable to these as well. To estimate the amount ofactivity remaining in the pulp, the pulp was extracted with methanol andthe methanol-soluble material was prepared in aqueous

At present, the MAW (methanol:acetic acid:water) extract is preferred,

                  TABLE VIII                                                      ______________________________________                                        Anti-Adhesion Activity of Selected Products During                            Preparation of Extract                                                        Sample                 Normalized activity                                    ______________________________________                                        1.  MAW initial extract    14.9                                               ppt. from initial extract                                                         1.2                                                                       3.  Prior to EtAc extraction                                                                             5.7                                                    (step 110, FIG. 1)                                                        4.  Step 114, aqueous phase                                                                              8.4     (16.8, 0.0)                                5.  Step 116 (evap. organic phase)                                                                       33.8    (117.7, 0.0)                               6.  EtAcEt.sup.1 10:1:5 initial extract                                                                  4.3                                                ppt. from initial extract                                                         3.9                                                                       8.  EtAcEt 5:1:10 initial extract                                                                        0.0                                                ppt. from initial extract                                                         7.7                                                                       10. Ethyl acetate initial extract                                                                        3.3                                                ppt. from initial extract                                                         6.4                                                                       12. Boiling water          6.1                                                ppt. from initial extract                                                         5.3                                                                       Step 1002 product                                                                 23.9                                                                      15. Refluxed water         4.1                                                ppt. from initial extract                                                         6.6                                                                       Step 1002 product                                                                 39.4                                                                      18. 10% Ocean Spray powder 4.6                                                19. Step 1001 wash discard 4.1                                                20. Step 1001, 2nd wash discard                                                                          0.0                                                21. Step 1002 product      34.5                                               22. CHCl.sub.3 /EtAc extract of 1002 prod.                                                               22.7                                               23. Step 1008A product     45.1                                               24. Step 1008B HCl eluate  0.0                                                25. Step 1008C methanol eluate                                                                           92.1                                               ______________________________________                                         .sup.1 EtAcEt = ethyl acetate/acetic acid/ethanol                        

as it appears to result in a greater recovery of activity in the initialextraction from berries and to leave a smaller proportion of activity inthe pulp.

FIGS. 11A-F are chromatograms on a C18 column of selected preparationsincluding some from Table VIII. FIG. 11A is a chromatogram of sample #17in Table VIII, which is a 10% solution of Ocean Spray powder. FIGS.11B,C are chromatograms of the water washes of the C18 column from step1000, corresponding to samples #18, #19 in Table VIII. FIG. 11D is achromatogram of the methanol eluate from step 1000 containing the activefraction and corresponding to sample #20 in Table VIII. In FIG. 11D agroup of 360 nm-absorbing peaks 1100 eluting at between 30 and 42minutes can be seen. This group of peaks is not detectable in FIGS. 11Bor 11C, and the relative amount of material eluting in these peaks ismuch less in the starting material (FIG. 11A). A group of elution peaks1102 absorbing at 512 nm, characteristic of anthocyanins, is also seenin FIG. 11D.

FIG. 11E is a chromatogram of a sample which has been further subjectedto cation chromatography, step 1008, corresponding to sample #22 ofTable VIII. Peaks 1100 are still present in amount similar to that ofFIG. 11D, but peaks 1102 (the anthocyanins) are considerably reduced inamount. Additionally, a group of small peaks 1104 eluting at about 23 toabout 27 minutes is detectable. FIG. 11F is a chromatogram of an HCleluate of the cation column, corresponding to sample #23. Peaks 1102 arepresent in substantial amount, while peaks 1100 are nearly absent.

Comparison of the RBC assay values and the chromatograms for the samplesof FIGS. 11A-11F reveals that the presence of significant activity inthe RBC assay is positively correlated with the presence of peaks 1100.

A sample of the product of step 1008 in the process of FIG. 10 wassubjected to HPLC on a large (50 ml) C18 column (FIG. 12). The indicatedfractions 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214 were collectedand analyzed in the RBC assay (Table IX). Since fractions 1200-1208 allcontained significant levels of activity, it appears that the extractmay contain multiple components contributing anti-adhesion activity.From the data it also appears that tannin or tannin-like compounds

                  TABLE IX                                                        ______________________________________                                        Comparison of Activity in Selected HPLC Fractions                             Sample      Normalized Activity                                               ______________________________________                                        1200        10.5                                                              1202        25.1                                                              1204        37.8                                                              1206        45.1                                                              1208        41.6                                                              1210         0.0                                                              1212         0.0                                                              1214         0.0                                                              ______________________________________                                    

contribute anti-adhesion activity to the extract. Tannins absorb UVwavelengths strongly (at 230 nm and 280 nm). Fractions 1200, 1202 and1204, which have little material absorbing at 360 nm, have significantelution peaks of material absorbing at 230 and 280 nm, in the wavelengthrange expected for tannins. It is known that tannins may be retained onlipophilic columns similar to the C18. Thus one might expect an extractproduced by the process to include tannin components. Portions offractions 1200, 1202, 1204, 1206, 1208 were re-run on analytic C18columns; the results are shown in FIGS. 13A-E.

As shown in the disclosed embodiments and Table VIII, an initial acidextraction of the berries or plant material solubilizes the activefraction. However, in an alternate embodiment, the plant material may befirst extracted with a basic solution (high pH) which will result inmuch of the active compounds being left in the pulp or residue. The pulpor residue itself is thus enriched for the anti-adhesion activity. Sucha "reverse" approach for enrichment of a substance known to beacid-soluble, is well known in the art. The pulp or residue can then befurther processed by one or more of the steps disclosed in FIGS. 1 and10 to obtain a more pure preparation of the active fraction. Theobserved property of acid solubility/base insolubility of the activefraction may also be exploited in other ways to produce an extract orcomposition highly enriched for the active fraction, as will be apparentto one skilled. For example, a sufficient amount of a base can be addedto a partially enriched acid extract to selectively precipitate theactive fraction.

A method of inhibiting the adhesion of bacteria to surfaces comprisesthe steps of providing a Vaccinium extract enriched for anti-adherenceactivity and for 360 nm-absorbing polyphenol compounds, and applying aneffective amount of a composition comprising the extract in anacceptable carrier, to a surface having bacteria such as E. coli adheredthereto to disengage the bacteria from the surface. Desirably, thesurface is rinsed to remove the disengaged bacteria. The method isuseful to inhibit the adhesion of bacteria to such surfaces as teeth,other bacteria adhered to teeth, to human oral epithelial cells, humanepithelial urinary tract cells; and to clean dental implants, bacterialfermentation vats, and the like.

The invention is described with reference to specific embodiments, plantspecies and parts, buffers and chemical procedures and the like.However, it will be recognized by those skilled in the art that varioussubstitutions can be made without departing from the spirit and scope ofthe invention. In particular, it is known that polyphenols includingflavenoids and anthocyanins can be isolated and/or partially purifiedfrom plant materials by a number of different methods. It will furtherbe recognized that these alternate methods, and consequent changes inother steps of the method including removal of sugars, of varioussolvents, and of anthocyanins from a composition comprising partiallypurified polyphenols, fall within the scope of the present inventedVaccinium extract.

What is claimed is:
 1. A composition for inhibiting the adhesion of E.coli bacteria to surfaces in a mammalian oral cavity, comprising:anextract prepared from whole cranberries and enriched by fractionating ona cation column to selectively enrich said extract with respect toflavonoid and polyphenol compounds so that the amount of anthocyanins insaid extract is less than about one-tenth the combined amounts ofpolyphenol and flavonoid compounds in said extract; and an aqueous oraqueous-alcohol liquid medium suitable for rinsing of a mammalian oralcavity.